PROJECT SUMMARY: Expansion of our understanding of hematopoiesis, including identification of key biochemical modulators, is critical to the development of novel interventions and improved potentially curative procedures such as bone marrow transplantation. While much study has occurred on the roles of kinases, the hematopoietic roles of the serine/threonine, multi-subunit PP2A family phosphatases, PP2A and PP4, have only begun to be examined. PP2Ac is reversibly methylated on its carboxy-terminal leucine (L309) by the methyltransferase, LCMT-1, and the methylesterase, PME-1, and this methylation regulates formation of certain PP2A complexes, and thus subcellular targeting and specificity. PP4, which is ~60% identical to PP2A, is also methylated on its carboxyl- terminal leucine. We recently found LCMT-1 is also the PP4 methyltransferase and selectively regulates certain PP4 complexes in addition to select PP2A complexes. Through the creation and initial study of a gene- trap LCMT-1 knockout (KO) mouse and subsequently a pan-hematopoietic conditional knockout (cKO) of the LCMT-1 allele, we show equally striking reductions in multilineage HSC competitive repopulation activity despite relatively normal adult HSC homeostasis. Therefore, LCMT-1 may have cell intrinsic roles in promoting HSC fitness uniquely during the stress at early times post-transplantation. This proposal is a multi-investigator application that leverages the biochemistry and experimental hematology expertise of both PIs. Together we have discovered that LCMT-1 plays a critical intrinsic role in hematopoiesis that is specific to HSCs in the post- transplantation expansion, survival, and self-renewal phase. In contrast, LCMT1 doesn't play a role during HSC maintenance and cycling during steady-state conditions. Because LCMT-1 substrates are limited to PP2A family phosphatases due to the unique way LCMT-1 must interact with both specific active site and C-terminal residues, we hypothesize that LCMT-1 promotes high levels of ?methylation-dependent? PP2A and PP4 heterotrimers that in turn modulate key regulatory phosphorylation sites on critical proteins such as Raf to promote mitogen-associated protein kinase (MAPK) pathway signaling required for the early proliferative drive during hematopoietic repopulation. In this proposal, we will test this hypothesis and begin dissecting mechanism by utilizing LCMT-1 cKO mice to define specific defects in adult HSC multilineage repopulating activity using flow cytometry-based assays. Assays will compare steady-state and replicative stress responses. The importance of LCMT-1 catalytic activity will be determined, the involvement of methylation-dependent PP2A complexes in repopulating function will be evaluated, and therapeutic rejuvenation strategies will be explored. Accomplishing these objectives will move the field forward by improving understanding of signaling regulation of hematopoiesis and by providing avenues for manipulation of LCMT-1 and PP2A family phosphatases to accelerate the rate of hematopoietic regeneration.